The proposed work involves density functional theory (DFT) calculations of geometric and electronic structure, electrostatics calculations, and quantum-mechanics/molecular-mechanics/molecular-dynamics (QM/MM/MD) simulations to provide a detailed mechanistic understanding of the catalytic reaction pathways and mechanisms of proton pumping in B-type and A-type cytochrome c oxidases (CcO's). Aim 1. Develop a quantum/electrostatic model explaining how chemical bonding and proton/electron flow to molecular oxygen within the Fea3-CuB dinuclear complex (DNC) leads to proton pumping across the membrane. QM/MM/MD studies will provide insights into dynamic processes of proton uptake, proton transfer within the DNC and the proton exit channel from the DNC. Aim 2. Detection and characterization of various oxygen species between and/or bridging the Fe-Cu species will be related to corresponding electronic states from DFT. Calculations of vibrational spectra and other electronic properties (Mssbauer) will be performed to connect our hypotheses with experimental spectroscopies. Aim 3. New single crystal X-ray structures at high resolution with little radiation damage will be obtained for B- type CcO from Thermus thermophilus. These will be analyzed in comparison to other structures and DFT calculations. Aim 4. We will further develop current methodologies to improve the quality of quantum chemical DFT calculations for these large active site models, to analyze dynamic processes with QM/MM/MD, and for the physical description of the remaining protein/membrane/aqueous solvent environment. CcO (Complex IV) of mitochondria links electron transfer through the electron transport chain to proton pumping across the inner membrane of the mitochondria, and similarly, across the plasma membrane in most aerobic bacteria. This is the proton motive force utilized for ATP production. Mitochondrial CcO's play an essential role in human health because adequate ATP supplies are required for most important metabolic functions. Also, disruptions in electron or proton transfer reactions or oxygen binding at CcO can lead the production of damaging reactive oxygen species including hydroxyl and superoxide radicals, and hydrogen peroxide. Understanding the structures, mechanisms, and functions of mitochondrial CcO's is important for better analysis of many genetic and metabolic diseases and cancers, and is also relevant to pathologies of aging.